Abstract The human APOBEC3 locus is composed of seven different cytidine deaminase proteins (A3A to A3H) that display antiviral activities against numerous viruses including HIV. Several of these host defense proteins, such as A3G, are incorporated into virions and target HIV by mutating the single stranded viral DNA during reverse transcription in the next cycle of infection. HIV, however, counteracts the antiviral activity of these APOBEC3 proteins by encoding the Vif accessory protein that leads to their proteasomal degradation in HIV producer cells. The antiviral activity of A3A remains unclear in contrast to the better-characterized anti-HIV functions of A3D, A3F, A3G and A3H. Of note, A3A is unique among the APOBEC3 cytidine deaminases because it targets both DNA and RNA. Moreover its expression is potently induced by Type I Interferon in myeloid cells such as macrophages and dendritic cells. Our preliminary findings using knockdown as well as overexpression of A3A show that A3A inhibits HIV directly in the target cell. We hypothesize that A3A acts as a lentiviral gatekeeper via a currently unknown mechanism. This hypothesis will be tested in three independent Specific Aims. In Specific Aim 1, we will characterize the impact of A3A overexpression on HIV infection using a panel of different HIV strains. Furthermore, we will perform CRISPR knockout experiments in myeloid cell lines and primary human cells to test the antiviral role of endogenously expressed A3A. In Specific Aim 2, we will dissect the mechanism of A3A restriction by defining at which step of the viral life cycle the block to infection occurs. We will also measure A3A-induced mutagenesis by deep sequencing of HIV proviral DNA. In Specific Aim 3, we will decipher the full spectrum of A3A transcript variation and protein expression in primary T lymphocytes and myeloid cells stimulated with different interferons. Lastly, we will use CRISPR transactivation to induce A3A expression in CD4+ T cells and determine the extent to which A3A renders these cells resistant to infection by different HIV strains. Collectively, the proposed studies will elucidate the anti-HIV activity of A3A and deliver mechanistic insights on how to exploit A3A to make susceptible human cells resistant to HIV.